1. Field of the Invention
The present invention relates to a method and equipment for the implementation of a biotechnological process ensuring that the process solution travels in a desired direction.
2. Description of the Prior Art
The field of biotechnology is currently marked by a strong trend of developing new technologies also for high-volume products, such as fuels, chemicals and bioplastics. Consequently, industrial biotechnology and microbiology are rapidly extending their main focus not only into enzymes, drugs and other products with a relatively low production volume but also into new areas of high interest. This development arises from such as concern over the availability and sufficiency of oil and other fossil fuels and their soaring prices, state of the environment and climate development. A positive impact on these issues can be achieved by a more wide-spread use and scope of industrial biotechnology. Biotechnology can also help us to avoid using many high-risk, dangerous or harmful chemicals.
In order to reach the objectives listed above, we will need new technologies for implementing biotechnical reactors, or bioreactors or fermentors, in a novel way, efficiently and optimally. Similarly, the downstream processing and recovery of products need to be developed. In order to reach optimal results in these tasks, or performing bioreactions in reactors and simultaneous or subsequent post-treatment processes, advanced measurement technology, process control and modeling are needed.
Biotechnology solutions typically build on biomass based raw materials, which often can be organic waste of various types. Plant biomass is another typical raw material. In this case, plants with high sugar or starch content can be used. On the other hand, the use of plant materials, such as cellulose, hemicellulose and lignins from wood materials has also been envisioned, and research and production projects of this type have already been realized. When using these macromolecules as sources of carbon and energy for microbial processes, it is usually necessary to hydrolyse them, or subject them to other types of physical, chemical or enzymatic treatment. This preliminary treatment enables an efficient use of the chemical energy bound in these materials for microbial growth and product formation. Lots of these plant-based macro-molecules or polymers are available in the nature, not to mention many waste materials. When using these, it may often be beneficial to add such as nitrogen components in the process or process solution as a source of nitrogen for the microbes, or various types of vitamins or other growth factors vital for the microbe in the process.
Other factors important for microbial growth and function include adjustments for a favorable physicochemical parameters of the process and for compensating changes caused by the microbe itself, thus eliminating any risks leading to drawbacks in the microbe growth, process or product formation. Various environmental factors that need to be taken into consideration in optimization include temperature, pH, partial pressures of oxygen and other gases, osmolarity, viscosity of the process solution etc. Many of these properties also play a role in the downstream processing and recovery of products.
Normally, a bio-reaction or fermentation takes place in a solution as a submerged cultivation: microbes take substances from the process solution, and the products are also formed in the solution. In case the product is the actual microbe cell material, this needs to be separated from the process solution and recovered using different types of downstream processing and purification methods. Microbe cells or enzymes, or other corresponding biocatalysts performing a reaction, may occur freely in the process solution. Often they may also be attached to a carrier material or carrier phase, i.e. be immobilized into or on that carrier. This often crucially extends the period of the biocatalyst usage and considerably increases its tolerance for various environmental pressures and stresses compared to a non-immobilized biocatalyst. In some cases, immobilization also facilitates the downstream.
When using microbes, the bioprocess often achieves an advantage if complex reaction pathways are avoided. Especially, when using microbes that assimilate or have the ability to fix atmospheric nitrogen, these advantages are highlighted. In some cases, biocatalyst properties can be improved by means of genetic manipulation, but in this case, close attention should be paid to health-related and ecological considerations. At best, the use of microbes considerably reduces the formation of harmful wastes.
Processes developed by the assistance of microbes are often called bio-refineries, especially when they are meant to complement or replace petrochemical industry. One benefit of these activities is that a number of products and processes can be run simultaneously. On the other hand, the same equipment can be used for different production and downstream processes. This may reduce the investment costs in biotechnical processes compared to those of chemical or petrochemical industries. However, additional costs in biotechnology may incur from demand for aseptics, which places high demands to the quality of materials, throughputs etc. Similarly, heterogeneity of the process solution may increase the costs significantly. If case waste is used as raw material, on the other hand, its negative price may have a positive effect on the total costs of the process, as well as multiple effects of positive environmental impacts or reductions in the emissions.
Often problematic in terms of the total result of a bioprocess is the fact that the product formation or production does not take place at a rate high enough. This is causing diluted process solutions or product mixtures, leading into high expenses in product recovery and purification. Similarly, in case the product is gaseous or a vaporizing substance and it can be separated by such means as distillation, the concentrations and production rate often remain low. In these situations, an adequate product concentration cannot be obtained sufficiently fast, which has a negative impact on the cost-effectiveness of the whole process. The risks of contamination by undesirable microbes and consequently great losses are increased. In order to optimize the entire process, it is often sensible to model it, allowing the simulation of the effects of various regulations on the reaction parameters in the process solution through the model. These regulations cannot, however, replace on-line measurements that yield accurate information on the process that is under way.
Placing different types of probes and sensors in the right areas in the bioreactor, or in an adequate number of locations in the various sections of the process solution, may emerge as a problem. In high-volume production plants, this may end up being highly expensive, and the sensor throughputs may cause contamination problems. Consequently, obtaining accurate data on the entire bioreactor or the situation of the whole process solution for modeling it and its biochemical regulations may be difficult. In order to resolve this technical issue, too, new types of bioreactors are needed. In the production of biofuels, biochemicals or other large-volume products, these problems are often emphasized, as is the fact that the high requirements placed on various materials in the bioprocess, such as steel quality, will increase the overall costs.
Many quantitative parameters in bioreactors are measured continuously. These may include monitoring not only the above-mentioned physicochemical values, such as temperature, pH and oxygen partial pressure, but also measurements of substrate and end product concentrations. These give important data on the progress of the process for its regulation, use of raw materials and product formation. With respect to these measurements, it is also essential to get a true picture of the whole process solution or bioreactor status.
Because bioreactor raw materials and end products usually are heterogeneous, a type of platform thinking in the biochemical, biotechnical and microbiological sense is useful. Certain organisms and their metabolic pathways offer an opportunity to vary the production, raw material use, production directions as well as the formation and recovery of different products. According to this way of thinking, the bioreactor or bioprocessor in a certain sense is an ecosystem subject to the laws of microbiology, which can also be utilized in a technological sense. The basis of all technological design and construction in macroscopic scale should lie in understanding of microscopic phenomena and observation of microbial interaction. What we need are new types of technological entities taking into account the phenomena occurring at the interface between the living biological material and engineering technology. These entities comprise of comprehensive measurement and regulation methods, production control taking into account the whole reactor volumes, more efficient movement of materials, and development of the recovery, and its integration into the production method, among other things. In this wider sense, developing bioreactors will be the most important task of bioprocess technology in the near future.
Various bioreactor and fermentor solutions include Stirred Tank Reactors with mechanical stifling, static fermentors, equipment for cultivation and production reactions on solid or semi-solid beds (e.g. techniques used for cultivating plant and animal cells), columns of various types (that may feature bubbles), air lift reactors, hollow fibre fermentors, basins and containers (with or without stirring), and bioreactors with immobilized biocatalysts (packed-bed or fluidized-bed types). In this application, the terms reactor, bioreactor and fermentor refer to equipment in which biochemical production or a reaction is performed. The fermentation or bioreaction may be of a fed-batch type or a continuous one. The former starts at a certain point, subsequently going through the whole process, mainly with the same raw materials; whereas in a continuous reaction, new raw materials are continuously fed in and correspondingly some part of the used materials removed. The problems inherent in the latter type of reactor, however, include a low level of utilization of the raw materials, even if some speeding up of the reaction is usually possible to accomplish e.g. in a chemostat type fermentor. Currently, majority of large-scale process solutions are of the so-called fed-batch type, in which the raw materials (nutrient medium for microbes or other cells) are added in a number of batches during the bioreaction into the original process solution (and the products thus formed often are also recovered in batches). This is naturally not applicable to acid or base additions used to regulate the fermentor or additions of other additives or regulators, but the addition of the actual main process raw materials only.
The objective of industrial microbiology and process biotechnology is to form the desired product, such as a cell mass, enzyme, chemical, polymer or fuel, by an energetically advantageous reaction in a bioreactor, making use of the biocatalysis of microbes or other cells (or filamentous growth forms) or the enzymes of the above-mentioned organisms. The biocatalyst may also be developed or refined through genetic recombination methods, in which case various safety considerations and risks should be taken into account with special care in the development of the biocatalysts, for example regarding the risks of unintentionally transferring genetic material. A part of a cell or cells may also sometimes work as a biocatalyst. What is essential is producing the desired products with lower amounts of reaction energy than in a corresponding chemical or physical reaction would be achievable. Bioreactors and biocatalysts may, in such cases as in polymerase industry, contribute to the production of substances and structures that cannot be achieved chemically by means of traditional synthesis. Similarly, by combining different microbes or organisms together (or with various enzymes) in the one and same reaction, process, or technological platform, such as in efficiently breaking down materials otherwise difficult to degrade. This phenomenon of accomplishing degradation as a result of the combined action of e.g. microbes, in a case which any individual microbe could not manage alone, is referred to as commensalism.
Cells that are used as biocatalysts are usually cultivated in the reactor until the process or reaction progresses up to a certain point. The cultivation may take place in one part of the reactor, or in case of reactors connected in a series, in some of the reactions, or during a temporary and limited period in the reaction. In any case, what often may become a problem is the fact that the growth of microbes or other cells requires different conditions from the formation of the actual product. Many antibiotics and enzymes, for example, are secondary metabolites, which can be formed after an active growth phase only. In this case, one must be able to prevent the formation—after the growth or other previous reaction or treatment phases—of components preventing or slowing down the actual product formation, also by the biocatalysts themselves. Contaminating microbes occurring in the raw material or emerging during the process can also in a more or less crucial manner impede the action of the biocatalyst, and also the growth of the actual process microbe and performance of production.
In terms of the overall cost-effectiveness of a bioreactor, it is also vital to ensure that the various nutrients are efficiently used and that the microbe's own control mechanisms, such as end product inhibition or catabolite repression, do not significantly reduce reaction efficiency. This can be influenced by using mixed instead of pure microbial cultures, or for example by removing substances potentially inhibiting, such as the end products or other products (such as metabolic wastes products), from forming into the process solution during the entire reaction or inside the reactor, as soon as these are formed. They can for example be recovered by means of sieving, filtering, two-phase fermentation, chromatography or vaporization as well as evaporation (e.g. distillation). In many techniques, vacuum suction can be used. Bacteria belonging to the species Escherichia coli are industrially very important. In their cultivation the use of a fed-batch reactor reduces the inhibitory acetate formation in so-called overflow metabolism, which does not support bacterial growth. The use of this reactor type also reduces catabolite repression compared to ordinary fed-batch cultivation.
An attempt can also be made to actively influence the regulatory genetic elements of microbes by adding certain extra nutrients or additives, or activating or inhibitory factors, to a specific cultivation or reaction phase. In a traditional bioreactor, this usually affects the whole reaction mixture, as stifling usually spreads the substance in question everywhere in the process solution. In fact, this kind of conduct may even take place during normal regulation of fermentation pH or temperature. The optimum temperature for growth, for example, may be different from the optimum one for the product formation. Similarly, e.g. the bacteria performing the 2,3-butanediol fermentation may grow fast in aerobic conditions, but the best yield of butanediol is achieved in micro-aerobic conditions.
In a biotechnical reaction, the impact of products separating from the catalyst on regulation systems and product formation is emphasized, especially in case their removal from the vicinity of cells or another catalyst is prolonged, for example because of diffusion restrictions of the substrate solution or compounds in it. Similarly, the impact of metabolic wastes that inhibit or slow down the action of the biocatalyst is often highlighted because of diffusion restrictions. They also have an influence on the cells' nutrients supply and/or substrate intake. The diffusion of various gases also plays an important role, such as in influencing the availability of oxygen to aerobic organisms. In addition to oxygen, other gases may also be highly significant (Hakalehto et al. 2007).
Of various organisms that are useful in biotechnology, spore-forming aerobic and anaerobic bacteria could be mentioned, such as representatives of the genus Bacillus in the aerobic group, and in the anaerobic group most importantly the ones belonging to the genus Clostridium, the latter of which include bacteria performing acetone-butanol fermentation, or butyric acid bacteria. Of the genus Bacillus, thermophilic bacteria are particularly important in many applications, as at high temperatures their metabolism, and thus also product formation, usually speeds up considerably. Facultative anaerobic bacteria, such as E. coli (mixed acid fermentation) and Klebsiella sp. or other enterobacterial species (carrying out 2,3-butanediol fermentation), methane producers and methylotrophic bacteria, various types of degraders, e.g. the genus Pseudomonas (aerobic), and many others also are of interest for the chemical and polymer industries. Of the yeasts, such as Candida sp. and Saccharomyces cerevisiae are commonly used for various types of fermentation, for example ethanol fermentation, and production of animal feed. Moulds and filamentous actinomycetes perform a number of special biocatalytic functions. They are, for instance, important producers of antibiotics, and they form many hydrolytic enzymes, which are used for the hydrolysis of macro-molecules in biomasses.
Various biotechnical modeling may focus on enzymatic kinetic reactions, as well as on mass transfer properties as regarding substrates, products, biocatalysts and wastes in the bioreactor and process solution. From the perspective of regulation, in order to prevent the formation of inhibitory by-product flows, it is important that the raw material in the reactor is converted into the desired products in its entirety. The properties of enzymes (and also cells) as biocatalysts can be listed as specificity, selectivity, specific speed of product formation, nutrient intake rate and stability of the biocatalyst. Understanding mass transfer properties of substances requires studies on the mechanics and hydraulic properties of liquids. The boiling temperatures and other physical and chemical properties of various substances must be determined. It is also important to know the temperature limits and optimums for growth and product formation of microbes taking part in the reaction, as well as the corresponding values for the pH, dissolved oxygen and other environmental parameters. By using means of fluorescence spectra, we can measure the concentrations of sugars, various ions, growth factors or carbon dioxide (e.g. for monitoring of the breathing of the cells), which can be used to collect important data for the running the process. Extending all these measurements to the whole reactor volumes and process solution, however, often becomes overwhelmingly expensive. It is also important to determine the heat transfer properties for the entire reaction e.g. by using a thermal camera system. Studies on heat transfer and gas diffusion conditions may yield information on such parameters as the predictable possibilities of biofilm formation in various parts of the reactor. Liquid flows and movements can also be simulated by computer modeling. The importance of determining all these process parameters is only increasing as the process is scaled up. In larger volumes, arranging heat regulation and even distribution of the various substances is more difficult than on the lab scale. The MFA procedure (“Metabolic Flux Analysis”) based on knowledge of all biochemical pathways could be used to take into account all metabolic pathways and transformations through modeling them.
In addition to modeling, appropriate measurements are essential in order to control the events on real time and in real-life conditions. Predicting or monitoring different biological phenomena in particular by only theoretical modeling may become an insurmountable obstacle, and even parallel or repeated measurements may produce widely variable results due to the special features of different biological materials. In this connection, we must take into account that process solutions or suspensions are not usually homogeneous microscopically, but contain cells and other particles, the behavior being a very challenging task to model. In terms of practical reactor design, a great degree of homogeneity in the process solution is highly important, at least during a specific process phase of particular importance. Extending the measurements into the entire reaction space may involve difficulties, and the same is true with the arrangements of throughputs, which as such haven an impact on what happens in the process solution, as well as increase contamination risk and bring up the costs.
When biotechnology is needed to produce chemical components of a relatively low value on a large scale, new technical solutions are required. In addition to measurements, modeling and arrangements for mass transfers, the most important task is organizing downstream processing and product recovery. This can often be integrated into the production process. The available methods include distillation, membrane filtration, sedimentation, sieving, centrifugation, evaporation, ultra-filtering and various chromatographic methods. These can be combined, and different products recovered in the same process.